Cancer stem cells are a self-renewal small cell group displaying pluripotent potency enabling the differentiation into various tissues and cells like general stem cells. So, with a small number of these cells, a tumor can be induced in an experimental animal model. Cancer stem cells are also highly resistant against chemotherapy and radiotherapy (B. M. Boman, M. S. Wicha, Cancer stem cells: A step toward the cure, J. Clin. Oncol. (2008) 26:2795-2799).
Cancer stem cells were first reported in acute myeloid leukemia, and later were found in general solid cancers including breast cancer, leading to the identification of solid cancer stem cells (D. Bonnet, J. E. Dick, Human acute myeloid leukemia is organized as hierarchy that originates from a primitive hematopoietic cell. Nat. Med. (1997) 3:730-737; M. Al-Hajj, M. F. Clarke, Self-renewal and tumor stem cells. Oncogene. (2003)23:7274-7284).
The cancer stem cell specific expression markers have been reported for the identification of cancer stem cells. Among them, CD133, a membrane protein, has been used as a marker to recognize and isolate cancer stem cells in brain tumor. It has been reported that a new tumor can be developed by transplanting about 100 CD133+ cells in a nude mouse (S. K. Singh, C. Hawkins, I. D. Clarke, et al, Identification of human brain tumor initiating cells. Nature (2004) 432:396-401). Another membrane protein CD44 was also proposed as a cancer stem cell marker. According to a previous report, the cells separated with CD44(+)/CD24(−)/Lineage(−) in breast cancer were grown into a xenograft tumor (M. Al-Hajj, M. S. Wicha, A. Bentino-Hernandez et al, Proc. Natl. Acad. Sci. USA (2003) 100:3983-3988). ALDH1 (aldehydrogeanse 1), the detox enzyme that can oxidize intracellular aldehyde is also a cancer cell marker (M. Magni, S. Shammah, R. Schiro. et al, Blood (1996) 87:1097-1103; N. A. Sophos, V. Vasiliou, Chem. Biol. Interact. (2003) 143-144:5-22). The activity of ALDH1 is important for isolating the population of cancer stem cells in lung cancer cell line (F. Jiang, Q. Qiu, A. Khanna et al, Mol. Cancer. Res. (2009) 7:330-338). It has been reported that the cells demonstrating high ALDH1 activity were highly self-renewal, which is the characteristics of cancer stem cells, and were actively differentiated in such cancers as breast cancer and lung cancer. The prognosis is poor in cancer patients having the tumor cells showing high ALDH1 activity (E. Charafe-Jauffret et al, Clin. Cancer. Res. (2010) 16: 45-55).
In spite of the discovery of all those cancer stem cell specific markers, the cancer stem cell specific network and mechanism have not been known yet. Thus, to prevent the recurrence and metastasis of cancer and to eliminate cancer completely, it is necessary to develop an anticancer agent that removes cancer stem cells by targeting the cancer stem cells having the characteristics of stem cells in addition to the current cancer treatment method targeting cancer cells.
TSPYL5 gene belongs to TSPYL (testis-specific protein, Y-encoded-like) family, which is highly expressed in breast cancer and is expected to play an important role in the carcinogenesis process of breast cancer (L. J. van't Veer, et al, Nature. (2002) 415:530-536). TSPYL5 is also over-expressed in lung cancer cell line, activates PTEN/AKT pathway, accelerates cell growth, and increases radiation resistance (E. J. Kim. et al., Biochem. Biophys. Res. Commun. (2010) 392(3):448-453). USP7 (deubiquitylation enzyme for p53 activation) has been reported as an interacting protein of TSPYL5. It has been found that TSPYL5 acts as an inhibitor of USP7, and thereby increases p53 degradation, which results in the poor cancer prognosis (M. T. Epping, et al., Nat. Cell Biol. (2011) 13(1):102-108). It has also been reported that TSPYL5 is associated with transcription factors of various genes including aromatase, which catalyzes the aromatization of estradiol from testosterone involved in post-menopausal breast cancer (Liu Ml, et al., Mol Endocrinol. (2013) 27(4):657-70).
Currently, the cellular physiological aspects of TSPYL5 gene and cancer stem cells have been largely identified, and a method using shRNA or siRNA to inhibit the functions of TSPYL5 protein involved in radiation resistance and cancer stem cell characteristics has been used. However, there is a still technical limit in developing an inhibitor of radiation-sensitive or radiation-resistant cancer stem cells.
Thus, the present inventors tried to establish a method to control cancer stem cells by identifying a specific amino acid residue and its variants involved in cancer stem cell characteristics and radiation resistance in TSPYL5 protein. In the course of the study, the inventors confirmed that the phosphorylation of threonine, the 120th amino acid residue of TSPYL5, was regulated by PETM/AKT and further succeeded in the construction of the mutant of the 120th residue threonine (T120) of TSPYL5, T120D- or T120A-TSPYL5. Then, cells were transfected with the constructed mutants. As a result, the present inventors confirmed that the wild-type TSPYL5 and T120D moved into nucleus and stayed there but T120A-TSPYL5 did not moved in nucleus and instead was only expressed in cytoplasm and accordingly did not bind to AKT. When the phosphorylation was not induced, ubiquitination of TSPYL5 protein was increased but SUMOylation was suppressed. Also, the expressions of the representative cancer stem cell markers ALDH1A1, ALDH1A3, and CD44 genes and proteins were reduced. In addition, the growth and metastasis of lung cancer cells were also reduced and sphere formation was suppressed. As explained hereinbefore, the inventors proved that the growth or metastasis of cancer cells or the growth of cancer stem cells could be suppressed by inhibiting the phosphorylation of threonine, the 120th residue of TSPYL5. At last, the present inventors confirmed that the TS120T peptide represented by SEQ. ID. NO: 43 comprising the phosphorylation region 120T and its phosphorylation analogue TS120D peptide represented by SEQ. ID. NO: 44 could be effectively used as an inhibitor for the growth or metastasis of cancer cells or the growth of cancer stem cells, leading to the completion of this invention.